Memory B cells from older people express normal levels of cyclooxygenase-2 and produce higher levels of IL-6 and IL-10 upon in vitro activation

Worldwide the elderly population is increasing. The elderly show deficiencies in immune function. B lymphocytes are essential elements of the immune system responsible for antibody production. This laboratory previously showed that activated human B cells isolated from young adults express cyclooxygenase-2 (Cox-2) and that Cox-2 is essential for optimal antibody responses. Recent data suggests that Cox-2 expression decreases with age in mouse bone tissue. There is no information regarding Cox-2 expression in B cells from older human subjects. We investigated the expression and activity of Cox-2 in naïve and memory B cells from older people. We show that B cells from older subjects show similar Cox-2 protein expression and activity, antibody production and proliferation compared to younger people. However, we found that activated memory B cells from older people produce higher levels of IL-6 and IL-10 compared to young adults. Therefore, the dysregulated cytokine production could contribute to immune senescence in the elderly.     

Activation of IL-4 and IL-6 secreting cells by antigen

The number, antigenic specificity and phenotype of cells secreting IL-4 and IL-6 in mice immunized with ovalbumin or keyhole limpet haemocyanin (KLH) in Freund's adjuvant (FA) was studied. The frequency of cells producing either of these cytokines began to rise 6 days post immunization, peaked at 11–14 days post-immunization, and fell to background by 21 days. The number of spleen cells secreting IL-6 was higher than the number producing IL-4 at all time points. Boosting elicited an anamnestic response characterized by a significant increase in the number of cytokine secreting cells within 4 days. Cytokine production was induced in multiple strains of normal mice, and was critically dependent on the use of Complete FA in addition to antigen. Immunization induced IL-4 and IL-6 production in vivo while ‘priming’ additional cells to release these cytokines when reexposed to soluble antigen in vitro. The latter response was antigen specific and was dominated by non-B/non-T cells. Those cells may serve to boost the immune response in cases of persistent or repeated antigenic challenge.     

Fine structure of Sertoli cells in three marine snails with a discussion on the functional morphology of Sertoli cells in general

Summary The fine structure of Sertoli cells in three marine prosobranch molluscs has been studied with light- and electron microscopy. Sertoli cells of prosobranchs are modified columnar epithelial cells that maintain continuous contact with the basal lamina and extend from it to the lumen of a testicular tubule. Spermatogenesis takes place between adjacent Sertoli cells, but a continuous layer of cytoplasm separates the spermatogonia from the basal lamina, thus restricting the basal compartment to spermatogonium mother cells. Substances traversing the basal lamina from the interstitial space must pass either through or between the Sertoli cells. However, between the cells, a permeability barrier composed of septate and desmosome-like junctions blocks the passage of substances, such as the tracer lanthanum nitrate. The basally-located nucleus is irregularly shaped with fine granular euchromatin and some peripheral heterochromatin; satellite karyosomes border the nucleolus. There is an extensive intracellular digestive system that is used effectively to phagocytize waste sperm and residual cytoplasm. Cytoplasmic processes of Sertoli cells penetrate throughout the germinal epithelium. In some prosobranchs that exhibit sperm polymorphism these processes must coordinate to bring together a clone of eupyrene sperm and a carrier sperm at a particular time in development. The only cytoskeletal elements available within the processes to generate such movements are microtubules.We propose that the term nurse cell, which has been used in the past to describe at least three different cell types, including Sertoli cells and apyrene sperm, be restricted to abortive oogonia that contribute to development of an oocyte.This paper was cited in a previous publication (Buckland-Nicks et al. 1982) under the title: A comparative investigation into the relationship between Sertoli cells, eupyrene and apyrene sperm in the testis of two marine snails     

Meiotic progression of rat spermatocytes requires mitogen-activated protein kinases of Sertoli cells and close contacts between the germ cells and the Sertoli cells

Progression of germ cells through meiosis is regulated by phosphorylation events. We previously showed the key role of cyclin dependent kinases in meiotic divisions of rat spermatocytes co-cultured with Sertoli cells (SC). In the present study, we used the same culture system to address the role of mitogen-activated protein kinases (MAPKs) in meiotic progression. Phosphorylated ERK1/2 were detected in vivo and in freshly isolated SC and in pachytene spermatocytes (PS) as early as 3 h after seeding on SC. The yield of the two meiotic divisions and the percentage of highly MPM-2-labeled pachytene and secondary spermatocytes (SII) were decreased in co-cultures treated with U0126, an inhibitor of the ERK-activating kinases, MEK1/2. Pre-incubation of PS with U0126 resulted in a reduced number of in vitro formed round spermatids without modifying the number of SII or the MPM-2 labeling of PS or SII. Conversely, pre-treatment of SC with U0126 led to a decrease in the percentage of highly MPM-2-labeled PS associated with a decreased number of SII and round spermatids. These results show that meiotic progression of spermatocytes is dependent on SC-activated MAPKs. In addition, high MPM-2 labeling was not acquired by PS cultured alone in Sertoli cell conditioned media, indicating a specific need for cell-cell contact between germ cells and SC.     

An immunogold evaluation of cortical actin reorganization in pubertal Sertoli cells in vitro after PMA treatment

The aim of the present study was to investigate stress fibers and cortical actin reorganization in pubertal Sertoli cells in vitro after PMA treatment. Actin was studied by means of immunogold labeling, using the 'Progressive Lowering of Temperature' technique (PLT). Actin rearrangement was evaluated by a quantitative analysis of the gold label distribution. Eight hours after addition of 10(-7) M PMA, rearrangement of cortical actin was minimal, but stress fiber perturbation was significant as shown by immunogold labeling distribution measurements. PMA-mediated F-actin reorganization and redistribution in non-neoplastic cells is discussed, since these phenomena have been closely linked with cell transformation.     

Densin is a novel cell membrane protein of Sertoli cells in the testis

Cell-cell interactions between Sertoli cells and germ cells are crucial for the maturation of germ cells in spermatogenesis but the structural and functional aspects of the interactions remain to be fully elucidated. Densin is a junction protein suggested to play a role in establishment of specific cell-cell contacts in the post-synaptic densities of the brain and the slit diaphragm of the kidney podocyte. In the present study, densin was discovered to be expressed in the testis of the man and the mouse. Expression of densin at the gene and the protein level was studied by using RT-PCR and Western blotting analyses, and the localization of densin was explored with immunofluorescence staining. RT-PCR and Western blotting analyses showed that densin is expressed at the gene and the protein levels. Immunofluorescence staining localized the expression of densin to the cell membranes of Sertoli cells suggesting that densin may be an adherens junction protein between Sertoli cells and developing germ cells. Densin is a novel testicular protein expressed in the cell membranes of Sertoli cells. Its functional role remains to be assessed.     

Ankrd7, a novel gene specifically expressed in Sertoli cells and its potential roles in Sertoli cell maturation

The somatic Sertoli cells play an essential role in testis determination and spermatogenesis by providing nutrition and structural support. In the current study, we report on the novel Ankrd7 gene that contains five ankyrin repeat domains. This gene was specifically expressed in Sertoli cells and was regulated in a maturation-dependent manner. Its expression was restricted to testicular tissue, and its mRNA could be detected in testes at as early as 14 dpp (days post partum) using RT-PCR analysis. In both testicular tissue sections and in vitro cultured Sertoli cells, the Ankrd7 protein was localized to the nucleus of the Sertoli cell. Immuno-histochemistry and immunocytochemistry investigations showed that the protein was detectable in testicular tissues at 20 dpp, at which time Sertoli cells were gradually differentiating into their mature cellular form. These results suggest that Ankrd7 is probably involved in the process of Sertoli cell maturation and in spermatogenesis.     

Calcitonin gene-related peptide indirectly inhibits IL-7 responses in pre-B cells by induction of IL-6 and TNF-alpha in bone marrow

Calcitonin gene-related peptide (CGRP) has inflammatory and immunoregulatory properties. CGRP directly inhibits IL-7 induced proliferation in developing B cells and also induces soluble factors that inhibit IL-7 responses. We identified 2 cytokines, IL-6 and TNF-alpha, induced by CGRP, that inhibit IL-7 pre-B cell responses. CGRP induction of IL-6 and TNF-alpha mRNA in long-term bone marrow cultures is transient and IL-6 or TNF-alpha inhibit IL-7 induced colony formation by 60%. When added with CGRP, colony formation is completely inhibited. TNF-alpha directly inhibits IL-7 responses in B220(+)/IgM(-) cells whereas IL-6 inhibits only colony formation with whole bone marrow. This suggests that the effect of IL-6 is mediated by other cells in the bone marrow. These results suggest that the indirect effect of CGRP on IL-7 depends in part on induction of IL-6 and TNF-alpha.     

Resveratrol treatment reduces expression of MCP-1, IL-6, IL-8 and RANTES in endometriotic stromal cells

Endometriosis is an inflammatory disease affecting reproductive-aged women. Immunologic disturbance, as well as inflammation, have crucial roles in the pathogenesis of endometriosis. In this study, we evaluated the effects of resveratrol treatment on expression of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), IL-8, and regulated upon activation, normal T cell expressed and secreted (RANTES) in endometrial stromal cells from patients with endometriosis compared with non-endometriotic controls. Thirteen eutopic (EuESCs) and nine ectopic (EESCs) endometrial stromal cells from endometriotic patients as well as eleven endometrial stromal cells from non-endometriotic controls (CESCs) were treated with resveratrol (100 μmol/L) or ethanol, and gene and/or protein expression of MCP-1, IL-6, IL-8 and RANTES was examined at 6, 24 and 48 hours following treatment in the cells from all origins. Resveratrol treatment significantly reduced gene and protein expression of MCP-1, IL-6, and IL-8 in EuESCs and EESCs compared with CESCs (P < .05-.001, P < .05-.001 and P < .05-<.01, respectively), and this reduction was more noticeable in EESCs than EuESCs (P < .05-<.001). Besides, resveratrol treatment significantly reduced RANTES protein expression in EESCs in all time intervals (P < .05). Resveratrol treatment significantly reduced the expression of MCP-1, IL-6, IL-8 and RANTES in EESCs.     

IL-6 Undergoes Transition from in vitro Autocrine Growth Factor toin vivo Growth Inhibitor of B Lymphoma Cells

IL-6 is a multifunctional cytokine involved in differentiation and proliferation of immune cells. Moreover, it has diverse effects on the proliferation of tumor cells in vivo and in vitro. Although stimulating cell growth of multiple myeloma cells, it inhibits the proliferation of B16 melanoma cells and lung cancer cells. B9.55 cells, B-cell lymphoma, are IL-6-dependent cells, definitely requiring exogenous IL-6 for growth. When the cDNA for IL-6 was transfected into B9.55 cells, they began growing in an autocrine pattern without exogenous IL-6. To investigate the effects of IL-6 on B9.55 lymphoma in vivo, IL-6-transfected B9.55 cells (B9.G7) or neotransfected B9.55 cells (B9.vec) were injected subcutaneously into syngeneic mice. Initially, B9.G7 outgrew B9.vec, but after 3 weeks, B9.G7 grew slower than B9.vec. In addition, 5 µg of recombinant human IL-6 was injected daily into the tumor site. Reduced tumor sizes of IL-6-treated rats, similar to those observed in mice which received B9.G7, indicated that IL-6 itself is the mediator of tumor regression. When B9.G7 cells were injected into the irradiated normal mice, tumor regression was released compared with the untreated normal control, suggesting that radiosensitive host components were involved in the regression of B9.G7 cell growth. However, the tumor regression of B9.G7 cells was not released in SCID mice. Histologically, B9.G7 tumor demonstrated severe necrosis and apoptotic cells with infiltration of host inflammatory cells. Above data indicate that IL-6 functions as an autocrine growth factor for B9.G7 cells in vitro, but behaves as an autocrine inhibiting factor in vivo. These contrasting effects of IL-6 on tumor cells in vitro and in vivo will be facilitative in understanding the interaction of cytokines and host immune systems.They have contributed equally to this work.     

The involvement of IL-6 and IL-8 in acute invasive gastroenteritis of children

The involvement of the proinflammatory cytokines, interleukin 8 (IL-8) and 6 (IL-6), was studied during the first 72 h of acute invasive gastroenteritis. Study population included 33 infants and young children aged six months to six years and seven age-matched controls. As a group, patients with acute invasive gastroenteritis had an increased serum level of IL-8 and IL-6 as compared with healthy controls (p < 0.002 and p < 0.001, respectively). Subjects were then divided into two groups based on stool cultures (proven and non-proven bacterial cultures). Patients with bacterial-proven acute invasive gastroenteritis tended to have increased IL-8 serum concentrations (p < 0.07) as compared with those with non-proven bacterial etiologies and IL-6 levels were only detected in subjects with positive bacterial cultures (p < 0.05). When dividing each sub-group into early and late blood drawing with respect to disease onset, no statistical differences were found in each group but subjects with bacterial-proven etiologies had significant higher IL-6 levels as compared with non-proven etiologies at the two time points (p < 0.019 and p < 0.015, respectively).In conclusion, the proinflammatory cytokines, IL-6 and IL-8, are involved in acute invasive gastroenteritis. The difference in IL-6, and to a lesser degree IL-8, between proven and non-proven bacterial etiologies, needs further investigation.     

Synthesis and transport of different sphingomyelin species in rat Sertoli cells

Cellular phospholipids of Sertoli cells from immature rats were labeled with [¹⁴C]-choline. Two sphingomyelin bands (SM1 and SM2) were identified by TLC. The incorporation of [¹⁴C]-choline over a 45 h period of incubation demonstrated that there are differences in labeling kinetics between SM1 and SM2. The subcellular location of SM1 and SM2 was investigated by accessibility to bacterial sphingomyelinase. The results showed the existence of two SM pools in Sertoli cells, but an equal cellular distribution of SM1 and SM2. SM2 is characterized by a relatively high content of unsaturated fatty acids. The inhibition of vesicular flow by monensin determines a decrease of about 60–70% in incorporation into SM1 and SM2, suggesting the existence of at least two sites of sphingomyelin synthesis. Pulse-chase and time-course experiments indicated a phosphatidylcholine SM precursor product relationship and differences in kinetic properties between SM1 and SM2. Resynthesis experiments showed that monensin had only a partial inhibitory effect on SM1 resynthesis, and a second sphingomyelinase treatment demonstrated that the resynthesized fraction reached the outer leaflet of the plasma membrane. The 60–70% inhibition of SM synthesis by monensin showed that the trans-Golgi cisternae and the trans-Golgi network are the most likely sites of bulk SM synthesis, and that about 15% of SM was synthesized in the cis/medial Golgi apparatus. Additionally the results indicated that plasma membrane SM synthase activity could be the site of about 15% of SM synthesis in Sertoli cells.     

人IL-6受体胞外区真核表达载体的构建及体外表达

目的构建人IL-6受体（IL-6R）胞外区真核表达载体,检测其在体外培养细胞中的表达。方法利用PCR扩增IL-6R胞外区,克隆到pcDNA3．1（＋）中,用双酶切、测序鉴定。重组质粒通过脂质体转染HL-60细胞,用G418进行筛选,利用Western印迹检测IL-6R蛋白表达。结果PCR扩增出1218bp的目的片段,双酶切和测序结果显示重组质粒正确。Western印迹结果显示转染细胞能够表达目的蛋白。结论成功构建了人IL-6R胞外区真核表达载体,并且能够在真核细胞中表达。     

The effect of gamma irradiation on potato microtuber production in vitro

The effects of low doses of gamma irradiation and potato (Solanum tuberosum L.) cultivar on the production of microtubers in vitro were investigated. Nodal segments from virus free explants of three potato cultivars (cv.) were placed on tuberization inducing medium and irradiated with 4 doses of gamma radiation (2.5, 5, 10, 15 Gy). Cv. Diamant produced the highest number of microtubers followed by Draga and Spunta. Irradiation of the explants with 2.5 Gy of gamma radiation led to a significant increase in the number of microtubers (38% increase over the control). Average weight of microtubers was not significantly influenced by low doses of gamma irradiation. Draga microtubers were the largest followed by Diamant and Spunta. Microtubers resembled mature tubers in shape (Spunta was oval and Draga and Diamant were spherical). Size of microtubers was crucial for sprouting in vivo. It is suggested that only microtubers larger than 5 mm in diameter (250 mg) be used to produce minitubers in vivo. Since 2.5 Gy is a low irradiation dose, it can be used to enhance tuberization in vitro without fear of genetic changes in the used cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Rat Sertoli and spermatogenic cells express a similar gene, and its product is antigenically related to an outer dense fiber-associated protein.

We have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfide-linked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein. Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5-min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis.     

Morphological and karyological studies on in vitro Sertoli cells of adult crab-eating macaques (Macaca fascicularis)

Sertoli cell cultures were obtained from isolated seminiferous tubules of adult crab-eating macaques (Macaca fascicularis). Cells were identified by their morphological characteristics and their capacity to produce and release in the culture medium 17β-estradiol and androgen-binding protein (ABP). Several cells were undergoing mitosis. Karyological analysis showed both diploid and tetraploid metaphases. Patterns of nuclear scission were also observed.